Project Summary Autism spectrum disorders (ASD) are estimated to affect more than 1% of children under 8 years of age in the US. These neurodevelopmental disorders are characterized by impaired social communication, restrictive interests and repetitive or stereotyped behaviors. There are frequent co-morbidities in ASD, including epilepsy and intellectual disability. ASD can be divided into two main categories based on differing causal factors. Syndromic ASD are caused by highly penetrant, single gene mutations or genetic copy number variants that impact clinically relevant genes, while idiopathic ASD result from more complex combinations of genetic and environmental factors. Despite decades of effort, no drugs are available for specific treatment of ASD, which remain a major area of unmet medical need. Drug discovery for ASD has been limited by a lack of knowledge of underlying disease biology. Recent progress in elucidating the genetic basis of ASD is providing a foundation for new therapeutic discovery approaches. Evidence for synaptic dysfunction in genetic models of syndromic ASD and the identification of mutations in genes encoding synaptic proteins in idiopathic autism suggest that alterations in neurotransmission represent a core pathogenic mechanism. Moreover, there is evidence for shared disease mechanisms among ASD, including syndromic and idiopathic forms. These lines of evidence support therapeutic discovery strategies aimed at identifying and targeting shared neurophysiological disease mechanisms in ASD. The overall objective of this proposal is to combine our Optopatch all-optical electrophysiology platform with a newly developed method to modulate gene expression (CRISPR interference) and to apply this system to parallel generation and neurophysiological characterization of multiple ASD genetic loss-of-function models. Optopatch is the only optogenetic system with compatible, genetically-encoded actuator and voltage sensor components, enabling light-mediated stimulation and precise recording of electrical activity in neurons with unprecedented throughput. CRISPRi achieves gene knockdown by targeting a transcriptional repressor hybrid protein, KRAB-dCas9, to a specified gene target via a complementary guide RNA. Efficient knockdown of multiple genes is achieved simply by changing the guide RNA. We will first optimize the CRISPRi/Optopatch system and then apply it to characterizing effects on neuronal activity and synaptic transmission in neuronal knockdown models for 20 ASD genes defined by loss- of-function mutations. Through this large-scale phenotyping effort, we propose to identify core disease mechanisms in ASD. If successful, the program will be positioned for Phase II research aimed at generating commercially valuable, validated phenotypic screening assays for ASD drug discovery. Validated assays will provide an attractive basis for discovery partnerships with pharmaceutical companies to discover and develop novel, mechanism-based treatments for ASD. Ultimately, this program stands to benefit the large population of ASD patients and their families and caregivers by enabling discovery of new treatments.